Disordered protein-based seeds for molecular clustering

ABSTRACT

A system and method for reversibly controlling clustering of proteins around an engineered multivalent nucleus is disclosed. The system and method utilize clustering, which may be controlled by light activation or deactivation. The system and method enable the spatiotemporal control of protein supramolecular assemblies, including liquid-like droplets under some conditions, and solid-like gels under other conditions. The system and method can be utilized for segregating or locally concentrating desired proteins and/or RNA in cells or cell lysate, which may be useful for protein purification purposes, or for assembling single or multiple membraneless bodies within specific sub-regions of the cells. These synthetically assembled bodies may recruit both transgenic and endogenic proteins and other biomolecules, thus can be linked to affect and even trigger a plethora of cellular processes, including both physiological and pathological (e.g., protein aggregation) processes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/467,362, filed Mar. 6, 2017, which is herein incorporated byreference in its entirety. In addition, the Sequence Listing filedelectronically herewith is also hereby incorporated by reference in itsentirety (File Name: PRIN-53102_ST25.txt; Date Created: Apr. 18, 2019;File Size: 13,878 bytes.)

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.HR0011-17-2-0010 awarded by Defense Advanced Research Projects Agency(DARPA). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Cellular function relies on coordinating the thousands of reactions thatsimultaneously take place within the cell. Cells accomplish this task inlarge part by spatio-temporally controlling these reactions usingdiverse intracellular organelles. In addition to classic membrane-boundorganelles such as secretory vesicles, mitochondria and the endoplasmicreticulum, cells harbor a variety of membrane-less organelles. Most ofthese are abundant in both RNA and protein, and are referred to asribonucleoprotein (RNP) bodies. Among dozens of examples include nuclearbodies such as nucleoli, Cajal bodies, and PML bodies, and cytoplasmicgerm granules, stress granules and processing bodies ((Mao et al.,2011), (Anderson and Kedersha, 2009), (Buchan and Parker, 2009),(Handwerger and Gall, 2006)). By impacting a number of RNA processingreactions within cells, these structures appear to play a central rolein controlling the overall flow of genetic information, and are alsoincreasingly implicated as crucibles for protein aggregation pathologies((Li et al., 2013), (Ramaswami et al., 2013)).

From a biophysical standpoint, these structures are remarkable in thatthey have no enclosing membrane and yet their overall size and shape maybe stable over long periods (hours or longer), even while theirconstituent molecules exhibit dynamic exchange over timescales of tensof seconds (Phair and Misteli, 2000). Moreover, many of these structureshave recently been shown to exhibit additional behaviors typical ofcondensed liquid phases. For example, P granules, nucleoli, and a numberof other membrane-less bodies will fuse into a single larger sphere whenbrought into contact with one another ((Brangwynne et al., 2009),(Brangwynne et al., 2011), (Feric and Brangwynne, 2013)), in addition towetting surfaces and dripping in response to shear stresses. Theseobservations have led to the hypothesis that membrane-less organellesrepresent condensed liquid states of RNA and protein that assemblethrough intracellular phase separation, analogous to the phasetransitions of purified proteins long observed in vitro by structuralbiologists ((Ishimoto and Tanaka, 1977), (Vekilov, 2010)). Consistentwith this view, RNP bodies and other membrane-less organelles appear toform in a concentration-dependent manner, as expected for liquid-liquidphase separation ((Brangwynne et al., 2009), (Weber and Brangwynne,2015), (Nott et al., 2015), (Wippich et al., 2013), (Molliex et al.,2015)). These studies suggest that cells can regulate membrane-lessorganelle formation by altering proximity to a phase boundary. Movementthrough such an intracellular phase diagram could be accomplished bytuning concentration or intermolecular affinity, using mechanisms suchas posttranslational modification (PTM) and nucleocytoplasmic shuttling.

Recent work has begun to elucidate the molecular driving forces andbiophysical nature of intracellular phases. Weak multivalentinteractions between molecules containing tandem repeat protein domainsappear to play a central role ((Li et al., 2012), (Banjade and Rosen,2014)). A related driving force is the promiscuous interactions (e.g.electrostatic, dipole-dipole) between segments of conformationallyheterogeneous proteins, known as intrinsically disordered protein orintrinsically disordered regions (IDP/IDR, which are typically lowcomplexity sequences, LCS). Hereinafter, the terms intrinsicallydisordered protein, intrinsically disordered region, an intrinsicallydisordered protein region are used interchangably. RNA binding proteinsoften contain IDRs with the sequence composition biased toward aminoacids including R, G, S, and Y, which comprise sequences that have beenshown to be necessary and sufficient for driving condensation intoliquid-like protein droplets ((Elbaum-Garfinkle et al., 2015), (Nott etal., 2015), (Lin et al., 2015)). The properties of such in vitrodroplets have recently been found to be malleable and time-dependent((Elbaum-Garfinkle et al., 2015), (Zhang et al., 2015), (Weber andBrangwynne, 2012), (Molliex et al., 2015), (Lin et al., 2015), (Xiang etal., 2015), (Patel et al., 2015)), underscoring the role of IDR/LCSs inboth liquid-like physiological assemblies and pathological proteinaggregates.

Despite these advances, almost all recent studies rely primarily on invitro reconstitution, due to a lack of tools for probing protein phasebehavior within the living cellular context. However, a growing suite ofoptogenetic tools have been developed to control protein interactions inliving cells. The field has primarily focused on precise control overhomo- or hetero-dimerization ((Toettcher et al., 2011), (Kennedy et al.,2010), (Levskaya et al., 2009)). But recent work suggests the potentialof optogenetics for studying intracellular phases, demonstrating thatlight-induced protein clustering can be used to activate cell surfacereceptors (Bugaj et al., 2013), as well as to trap proteins intoinactive complexes ((Lee et al., 2014), (Taslimi et al., 2014)).

Thus, a platform which can be used to dynamically modulate intracellularprotein interactions, enabling the spatiotemporal control of phasetransitions within living cells is highly desirable. This platform couldalso be used for various biotechnological applications, includingprotein purification.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a platform for reversibly andnon-reversibly generating liquid droplets, gels, or protein aggregatesinside and outside cells by using nucleation cores, which may becontrolled by light. In the present invention, systems and methods areprovided for a system of protein constructs which may utilize aphoto-activatable or photo-deactivatable interaction between a lightsensitive receptor protein on a first protein construct and its cognatepartner on a second protein construct in order to control therecruitment of intrinsically disordered proteins onto cores comprised ofself-assembling protein subunits (see, e.g., FIG. 4). In this process,self-assembling protein subunits which are part of the first proteinconstruct will self-assemble and form a “core”. As each of theself-assembling protein subunits are fused to a light sensitive receptorprotein, light may be used to trigger the assembly or possiblydisassembly of a structure comprising the light sensitive receptorprotein on a first protein construct with a cognate partner of the lightsensitive receptor protein on a second protein construct, where thecognate partner is fused to a full length or truncated low complexity orintrinsically-disordered protein (see, e.g., FIG. 5).

Among the many different possibilities contemplated, the self-assemblingprotein subunit could be a ferritin heavy chain, and the intrinsicallydisordered region (IDR) can be the N terminal domain of FUS protein.Photo-inducible reversible heterodimerization between theself-assemblying units (e.g., part of the first protein construct) andIDR units (e.g., part of the second protein construct) could utilize,e.g., the engineered blue light sensitive receptor protein iLID and itscognate partner, sspB. One or both of the protein constructs may beadvantageously attached to a fluorescent protein marker. It iscontemplated that these protein constructs will be configured such thatafter being introduced into a living cell, exposing the living cell tocertain wavelengths of light will induce molecules within the livingcell to cluster or nucleate liquid phases, gels, or aggregates includingpathological protein aggregates such as amyloid fibers. In embodimentswhere photo-activation (or deactivation) is not required, it iscontemplated that phase separated clusters would be present in cellsindependent of the presence or absence of light.

The rapid and reversible clustering capabilities of the platform couldbe exploited for protein purification applications. For that purpose, atarget protein intended for purification is fused through a cleavableprotein tag to one of the protein constructs, preferably to the IDRcontaining construct. Transiently inducing clustering byphoto-activation will locally enrich target proteins in separate phases.Exploiting the distinctive physical and chemical properties of thesephases, for instance density, enables easy and rapid purification, forinstance by droplets sedimentation via centrifugation and supernatantremoval. Following a first such purification process, the target proteincan be cleaved out using specific protease, while the remaining clusterforming constructs are to be removed through a similar secondpurification process. Further, by selecting certain IDRs, the platformmay be configured to allow enrichment of proteins other than one boundto the construct having a light activated protein or their cognatepartners, allowing purification of target proteins which are notdirectly linked to one of the constructs.

Further envisioned is the formation of synthetic organelles by directlyimmobilizing several enzymes around a self-assembling core comprisingthe second protein construct and/or indirectly recruiting enzymes intothe phase separated environment generated by the self-assemblingintrinsically-disordered-proteins modified cores. This means of locallyconcentrating enzymes may also facilitate catalytic turnover forbiosynthesis and biodegredation applications. The method may alsoadvantageously control accessibility of a reactant by tuning solubilitywithin an encapsulating liquid phase.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A, 1B and 1C illustrate embodiments of a platform.

FIGS. 2A, 2B, and 3 are flowcharts of methods utilizing the platform.

FIGS. 4 and 5 are graphical depiction of the method.

FIG. 6 is a series of images illustrating the recruitment endogenousproteins around cores over time.

FIG. 7 is a graph illustrating the kinetics and reversibility of theplatform.

FIGS. 8A and 8B are images of a cell before and after certain areas ofthe cell were irradiated.

FIG. 8C is a graph illustrating the concentration capability of theplatform.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in further detail, it is to beunderstood that the invention is not limited to the particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, a limitednumber of the exemplary methods and materials are described herein.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise.

FIGS. 1A and 1B depict generalized embodiments of the disclosedplatform. The platform (10) generally comprises two types of proteinconstructs (12, 14).

The first protein construct (12) comprises at least one light sensitivereceptor protein (20) fused to a self-assembling protein subunit (30),which may be an oligomeric protein subunit. Optionally, a fluorescentprotein tag (25) may be included, either as indicated in FIG. 1 or otherlocations if desired. (25).

The at least one light sensitive receptor protein (20) may comprise oneor more similar or different proteins responsive to at least onewavelength of light, preferably a wavelength of light in the near UV,visible or infra-red regions, which are from about 350 nm to about 800nm. In preferred embodiments, the light sensitive protein is theengineered protein iLID, which consist of a modified LOV2 domain fusedat its C terminus to an ssrA peptide. In certain embodiments, theself-assembling protein subunit is fused to two or more LOV2-ssrAproteins. However, other light sensitive proteins may also be utilized,including Cry2, PhyB or a LOV2 domain fused to a signaling peptide otherthan ssrA. The self-assembling protein subunit (30) can be any proteinthat self-assembles, including but not limited to ferritin light chains,ferritin heavy chains, glutamine synthetase, and viral capsid structureproteins, or synthetic engineered self-assembling proteins. Onepreferred embodiment utilizes ferritin heavy chain subunits, which arecapable of self-assembly into a 24 mer complex with a spherical shellstructure. Assembled ferritin form deposits of iron-oxide at itsinternal cavity. By performing certain mutations, such deposits canbecome ferrimagnetic, thereby making modified ferritin responsive tomagnetic field.

The optional fluorescent protein tag (25) can comprise any appropriatefluorescent protein tag, such as mCherry, although the use of otherfluorescent proteins is also envisioned, including but not limited toGFP variants.

The second protein construct (14) comprises at least one cognate partner(40) of the light sensitive receptor protein (20), fused to a fulllength or truncated low complexity sequence (LCS) or IDR (60). As shownin FIG. 1B, the second construct (14) may also optionally comprise acleavage tag (70) or a target protein (80). Optionally, a fluorescenttag (50) may be included, either as indicated in FIG. 1B (between thecognate partner and the LCS or IDP) or other locations if desired.

The cognate partner of the light sensitive receptor protein (40) is anyappropriate cognate of the light sensitive receptor protein (20), whichmay include but is not limited to ssrB, Zdk, CIB, or PIF for LOV2-ssrA,LOV2, Cry2, or PhyB respectively. In preferred embodiments, the secondprotein construct comprises an IDP (60), which include but not limitedto full length or truncated forms of FUS [SEQ ID NO.: 1], DDX4 [SEQ IDNO.: 2], and hnRNPA1 [SEQ ID NO.: 3]. In some embodiments, the IDRcomprises amino acids 1-214 of FUS, 1-236 of DDX4, or 186-320 ofHNRNPA1. The fluorophore (50) can comprise any appropriate fluorescenttag, such as mCherry, although the use of other fluorescent proteins isalso envisioned, including but not limited to GFP variants.

When cleavage tags are utilized, at least one cleavage tag (70) istypically inserted between the functional region and a protein (80) thathas been targeted for, e.g., purification. A wide variety of cleavagetags are envisioned, including but not limited to: self-cleaving tags,Human Rhinovirus 3C Protease (3C/PreScission), Enterokinase (EKT),Factor Xa (FXa), Tobacco Etch Virus Protease (TEV), and Thrombin (Thr).

Variants of the two types of protein constructs (12, 14) canconcomitantly be used, for example, to allow multi-wavelengthsensitivity or functionalizing core proteins with different IDRs and/orenzymes.

In cases where photo-sensitivity is not necessary, rather than using twoconstructs to create a photo-activatable or photo-deactivatable systems,a single protein construct (16) may be utilized. As shown in FIG. 1C,the single protein construct could comprise a self-assembling proteinsubunit (30) and a full length or truncated low complexity sequence(LCS) or IDR (60). Optionally, the single construct could also includeat least one of a flouorescent tag (50), a cleavage tag (70), and/or atarget protein (80). For example, the single construct (16) couldcomprise a Ferritin protein fused to the FUS IDR. In this manner, thesystem could generate a disordered protein-based seed for molecularclustering without requiring photo-activation or -deactivation.

FIG. 2A depicts a flowchart of a method (100) for inducing clusters,which may be used for, e.g., protein purification. FIG. 2B shows theanalogous method (101) when utilizing a single non-activatable construct(16). The method generally comprises at least nine steps (six for thesingle construct). The first step is providing (110) DNA encoding firstand second protein constructs (12, 14) as described above, where thesecond protein construct (14) also encodes a target protein (see, e.g.,FIG. 1B, element 80). Alternatively a single light insensitive construct(16) encoding self-assemblying subunit, IDR, and a target protein can beused (see, e.g., FIG. 1C). The DNA encoding the constructs (12 and 14,or 16) is introduced (120) into living cells. Cells are grown (122) andprotein production is induced until cells reach desirable density. Cellsare then lysed (124) and if photo-activatable or -deactivatableconstructs are utilized, the lysate is, e.g., centrifuged (126) toremove larger cell debris. If photo-activatable or -deactivatableconstructs are utilized, supernatant is then exposed to at least onewavelength of light (130) that the light sensitive proteins areresponsive to, which induces molecules previously within the living cellto cluster or uncluster. As discussed above, the wavelength of light ispredetermined, based on the specific wavelengths to which the lightsensitive protein utilized in the constructs is responsive. It is notedthat for increasing yield, clustering by photoactivation may be appliedprior and during the cell lysis step, during which both self-assemblyingproteins and target protein constructs are still highly concentratedinside the cells. It is also noted that clustering may also confineadditional target proteins or RNA molecules, which are not directlylinked to the second protein construct, but help solubilize in theassembled phase and therefore can be purified even in the absence of thecognate partner of the light activatable protein.

The induced clusters may then be separated (131), typically viacentrifuge or using a magnetic field, in order to remove, e.g., theunclustered phase. The pellet is resuspended and followed by a cleavagestep (132), where the target protein is cleaved from, for example, theIDR (60). If photo-activatable or -deactivatable constructs areutilized, then after cleaving, a second induction step (134) is utilizedconcomitantly with removing clusters (136) via centrifugation or anapplied magnetic field in order to induce clustering of the coreconstruct (12) and the truncated cognate construct, thus leaving thetarget protein concentrated in the supernatant, which is then able to becollected. If a single construct is used, the second induction step(134) is not utilized, but the clusters are removed (136) following thecleaving step (132).

As shown in FIG. 3, the method (102) can be further modified. Forexample, the method can be modified to form intracellular syntheticorganelles (160) by either directly immobilizing several enzymes of arelated biochemical pathway around the core of self-assembling proteinsubunits comprising a plurality of first protein constructs (12), and/orby indirectly recruiting such enzymes into the phase separatedenvironment generated by the self-assembling LCS or IDR modified cores.For the former, a subset of the self-assembling protein subunits may befused to enzymes, while another subset are fused to light-activatableproteins, or fusions of the self-assembling protein subunits to bothenzymes and light-activated proteins may be utilized. For the latter,enzymes may be recruited to the phase separated environment throughinteractions mediated through, e.g., fusion with peptides/proteins thatpromote interactions with components of the condensed phase (e.g.,fusion of an enzyme to a segment of the FUS IDR, or to an engineeredpeptide designed to target the enzyme to the condensed phase).

The method and system can also be used to facilitate catalytic turnoverupon photo-activation by locally concentrating enzymes in specializedbiochemically reactive compartments inside or outside cells (170), forinstance for intracellular production of biofuels. And the method canalso be used to control accessibility of a reactant by tuning solubilitywithin an encapsulating liquid phase comprising theintrinsically-disordered protein. In preferred embodiments, theconcentration (170) follows inducing an additional protein—one not boundto the second construct—to cluster.

As these constructs are modular, properties can be varied, includingactivation/deactivation times, wavelength sensitivity, core size, lightsensitive receptor protein density on the core, IDR sequences, andreversibility.

FIG. 4 provides a graphical depiction of the method. In thenon-activated configuration (210), a core can be seen, comprising theself-assembling protein subunits (212) of a number of first proteinconstructs, each self-assembling protein subunit (212) fused to a lightsensitive receptor protein (214). One example of a light sensitivereceptor protein is a LOV2-ssrA domain. The second protein constructsremain unbound from the core, each second protein construct comprisingthe cognate partner (216) of the light sensitive protein (here, thecognate partner of the LOV2-ssrA domain is sspB) fused to full length ortruncated low complexity or intrinsically-disordered protein (e.g., FUS,etc.).

In the active state (e.g., upon photoactivation), the light sensitivereceptor protein (214) binds to the cognate partner (216) of the lightsensitive receptor protein. In this example, the buried ssrA peptidesbecome uncaged. Exposed ssrA rapidly bind their cognate sspB partners.Because the cognate partners (216) are bound to an LCS/IDR, theclustering of LCS/IDR around the self-assembled core leads to theformation of a photo-stabilized liquid droplet (220).

The phase-stabilized liquid droplet (220) may continue to grow to alarger phase-stabilized liquid droplet (230) by recruiting singlemolecules, such as additional second constructs (232), or endogenousLCS/IDRs (234, 262) or other proteins not fused to a cognate partner.And as shown in FIG. 5, the phase-stabilized liquid droplet (220) mayalso continue to grow via addition of single proteins (262), single coreparticles (264), or coalescence of mature multi-core particles (266).

An example of recruitment of FUS proteins by core based droplets can beseen by utilizing a first construct comprising ferritin fused to twoiLID-ssrA domains, and a second construct comprising FUSn fused tomCherry and sspB. In addition, the system utilizes a full length FUSfused to Cyan Fluorescent Protein (CFP). In this system, as shown inFIG. 6, significant mCherry fluorescence is seen as early as 15 secondsafter activation, and CFP fluorescence near the core based dropletsstarts to appear around that time. As the core droplets grow andcoalesce, CFP continues to be recruited, and after 15 minutes ofirradiation, significant CFP recruitment can be seen around the cores.

In this example, DNA fragments for human FUS, FUSn (residues 1-214) andhuman ferritin (heavy chain) were amplified by PCR using HeLa cell'stemplate cDNA. DNA fragments for iLID-ssrA, an engineered protein thatis based on Avena Sativa LOV2 domain fused to E. coli ssrA peptide, andE. coli sspB were amplified by PCR (Phusion® high-fidelity DNApolymerase, ThermoFisher Scientific) using PLL7.0:Venus-iLID-Mito (Addgene #60413) and PLL7.0:tgRFPt-SSPB WT (Add gene #60415) respectively. Anuclear localization signal from Gallus gallus ferritoid (18 aa encoding54 bases) was fused to the N terminus of iLID by sequential PCRs.

pHR:NLS-iLID-GFP-ferritin, pHR:NLS-iLID-iLID-GFP-ferritin,pHR:FUS(1-214)-mCherry-sspB, pHR: mCherry-sspB, pHR:FUS-CFP, pHR:DDX4(1-236)-mCherry-sspB and HNRNPA1(186-320)-mCherry-sspB plasmids wereconstructed using lenti viral pHR backbone through In-Fusion cloning ofmultiple inserts (In-Fusion® HD Cloning Plus HD cloning kit, Takara BioUSA).

To produce stable cell lines, lentiviral constructs were transientlytransfected with FuGENE® HD transfection reagent (Promega), followingthe manufacturer's recommended protocol, into HEK293T cells. Viruseswere harvested 48 hr after transfection and passed through a 0.45-μmfilter to remove cell debris. NIH 3T3 cells plated at ˜30% confluency in6-well dishes were infected with NLS-iLID-GFP-ferritin orNLS-iLID-iLID-GFP-ferritin containing viruses by adding 1 mL of filteredviral supernatant directly to the cell medium. Viral medium was replacedwith normal growth medium 24 hr after infection. A second construct,FUS-mCherry-sspB or mCherry-sspB, was subsequently added to the ferritinexpressing cells following two cell passages.

35-mm glass-bottom dishes were coated for 20 min with 0.25 mg/mlfibronectin and then washed twice with phosphate buffered saline (PBS pH7.4, Thermo). Cells were plated on the fibronectin-coated dish and growntypically overnight in normal growth medium to reach ˜50% confluency.All live cell imaging was performed using 60× oil immersion objective(NA 1.4) on a Nikon A1 laser scanning confocal microscope equipped witha stage top incubator (okolab) set to 37° C. and 20% CO₂.

For global activation, cells were irradiated with a 488 nm laser whileimaging was conducted using two wavelength (488 nm for GFP-ferritinbased constructs and 560 nm for mCherry-sspB based constructs). Forexecuting an activation-deactivation cycle, we typically used a 30-120 sdual wavelength excitation for iLID activation and GFP and mCherryimaging, which was followed by 2-5 min of 560 nm imaging for iLIDdeactivation. For recruitment assay, iLID was activated simultaneouslywith FUS-CFP imaging using 450 nm laser excitation, while mCherry wasimaged using 513 nm laser excitation.

For local activation, cells were excited by setting the 488 nm laser toscan a confined spherical or line-shape ROIs (0.3-1 μm indiameter/width), while imaging was conducted through the mCherryexcitation/readout channel only. Local activation for Fluorescencerecovery after photobleaching (FRAP) experiments was conducted byscanning a ring-shape region of interest (ROI) with 488 nm laser, whilebleaching of mCherry was performed at the center pixel of the ring usinga 560 nm laser.

FIG. 7 illustrates this platform's kinetics and reversibility. FIG. 7depicts the standard deviation (Std) of mCherry fluorescence intensityof the IDR-sspB construct over a number of activation and deactivationcycles. Change in Std indicates spacial redistribution of fluorophoresfrom a uniform siffusive state to a nonuniform clustered state. Forthis, a first construct comprising Ferritin fused to LOV2-ssrA and asecond construct comprising FUSn fused to sspB. In this example,droplets were observed after is of activation, and dissembling afterless than 1 min with an exponential decay half-life of about 11 sec. Asindicated in FIG. 6, the Std of Fluorescence indicates rapid kinetics,and a high degree of reversibility. When two or more LOV2-ssrA domainswere utilized, however, irreversible droplets were seen after threecycles of activation and deactivation.

FIGS. 8A-C illustrate some of the potential of the droplet's ability toconcentrate, e.g., the disclosed protein constructs in a singleintracellular cluster. FIG. 8A shows a cell comprising one example of aparticular system before being irradiated. This system utilized a firstconstruct comprising Ferritin fused to LOV2-ssrA and a second constructcomprising FUSn fused to mCherry and sspB. Rather than irradiating theentire cell, specific small areas of the cell were irradiated, each areaapproximately 1 micrometer in diameter. FIG. 8B shows the patternedactivation of multiple single droplets within the cell, each brightdroplet indicating a region where the proteins are concentrated. FIG. 8Cshows the mean mCherry intensity over time for the particular system,which is an relative measure of protein concentration. As shown in FIG.8C, the droplets formed were 100-fold more concentrated thannucleoplasm.

Kits may also be provided to simplify the use of these methods. The kitswill generally include plasmids for the two protein constructs (12, 14)or a single construct (16) as described above, as well as at least onelight emitting device that can be used to activate or deactivate thelight sensitive proteins. Kits may also include a microfabricated devicefor activation and collection of condensed liquid phases.

Thus, specific devices and systems for nucleated protein clustering havebeen disclosed. It should be apparent, however, to those skilled in theart that many more modifications besides those already described arepossible without departing from the inventive concepts herein. Theinventive subject matter, therefore, is not to be restricted except inthe spirit of the disclosure. Moreover, in interpreting the disclosure,all terms should be interpreted in the broadest possible mannerconsistent with the context. In particular, the terms “comprises” and“comprising” should be interpreted as referring to elements, components,or steps in a non-exclusive manner, indicating that the referencedelements, components, or steps may be present, or utilized, or combinedwith other elements, components, or steps that are not expresslyreferenced.

What is claimed is:
 1. A system, comprising: a first protein constructcomprising at least one self-assembling protein subunit fused to atleast one light-sensitive receptor protein capable of binding to acognate partner; and a second protein construct comprising the cognatepartner of the light-sensitive receptor protein fused to a full lengthor truncated low complexity or intrinsically-disordered protein region.2. The system according to claim 1, wherein the self-assembling proteinsubunit is ferritin.
 3. The system according to claim 2, wherein theferritin is a ferritin heavy or light chain.
 4. The system according toclaim 1, wherein the at least one light-sensitive receptor proteincapable of binding to a cognate partner is an engineered protein.
 5. Thesystem according to claim 4, wherein the engineered protein is iLID. 6.The system according to claim 4, wherein the self-assembling proteinsubunit is fused to two iLID proteins.
 7. The system according to claim1, wherein the light-sensitive receptor protein capable of binding to acognate partner is ssrA and the cognate partner of the light-sensitivereceptor protein is sspB.
 8. The system according to claim 1, whereinthe intrinsically-disordered protein region is selected from the groupconsisting of FUS or FUSn.
 9. The system of claim 1, wherein thelight-sensitive receptor protein capable of binding to a cognate partneris sensitive to at least one visible wavelength of light.
 10. The systemaccording to claim 1, wherein the second protein construct furthercomprises a fluorescent tag.
 11. A cell line that produces the system ofclaim
 1. 12. A method for forming an assembled phase, comprising thesteps of: producing at least one self-assembling protein subunit fusedto at least one light-sensitive receptor protein capable of binding to acognate partner; producing a cognate partner of the at least onelight-sensitive receptor protein fused to a full length or truncatedintrinsically-disordered protein region and a target protein; forming atleast one assembled phase comprising the at least one self-assemblingprotein subunit fused to the at least one light-sensitive receptorprotein and the cognate partner of the at least one light-sensitivereceptor protein fused to the full length or truncatedintrinsically-disordered protein region and the target protein, whereinthe at least one light sensitive receptor protein is bound to thecognate partner by exposing the at least one light sensitive receptorprotein to at least one predetermined wavelength of light.
 13. Themethod according to claim 12, wherein the formation of the assembledphase occurs within a living cell comprising pathological proteinaggregates.
 14. The method according to claim 13, wherein thepathological protein aggregates comprise amyloid fibers.
 15. The methodaccording to claim 12, further comprising separating the at least oneself-assembling protein subunit fused to the at least onelight-sensitive receptor protein bound to the cognate partner of the atleast one light-sensitive receptor protein fused to the full length ortruncated intrinsically-disordered protein region and the target proteinvia at least one of a centrifuge or a magnetic field.
 16. The methodaccording to claim 12, wherein the cognate partner of the at least onelight-sensitive receptor protein fused to a full length or truncatedintrinsically-disordered protein region and a target protein includes acleavage tag between the target protein and the intrinsically disorderedprotein region.
 17. The method according to claim 16, wherein thecleavage tag is selected from the group consisting of: Human Rhinovirus3C Protease (3C/PreScission), Enterokinase (EKT), Factor Xa (FXa),Tobacco Etch Virus Protease (TEV), and Thrombin (Thr).
 18. The methodaccording to claim 16, wherein the cleavage tag is a self cleaving tag.19. The method according to claim 12, further comprising the steps of:cleaving the target protein from the intrinsically-disordered proteinregion.
 20. A kit for inducing in vivo liquid-liquid phase separation,comprising: a plasmid encoding for a first and second protein construct;and at least one light emitting device configured to activate at least aportion of the first protein construct, wherein the first proteinconstruct comprising at least one self-assembling protein subunit fusedto at least one light-sensitive receptor protein capable of binding to acognate partner, and wherein the second protein construct comprises thecognate partner of the light-sensitive receptor protein fused to a fulllength or truncated intrinsically disordered protein.